GIPs, a family of polypeptides with transcription factor activity that interact with Goodpasture antigen binding protein

ABSTRACT

The present invention provides isolated GPBP-interacting 90 and 130 kDa polypeptides, and portions thereof (GIP90/130 polypeptides), antibodies to the GIP90/130 polypeptides, and pharmaceutical compositions thereof. The present invention also provides isolated GIP90/130 nucleic acid sequences, expression vectors comprising the nucleic acid sequences, and host cells transfected with the expression vectors. The invention further provides methods for detecting the GIP90/130 polypeptides or nucleic acid sequences, methods for inhibiting interactions between GPBP and GIP90/130 polypeptides, between pol k76 and GIP90/130 polypeptides or aggregation of GIP90/130 polypeptides, and methods for treating patients with autoimmune disorders or cancer.

CROSS REFERENCE

This application is a continuation of U.S. Utility patent applicationNo. 10/309,851, filed Dec. 4, 2002, now U.S. Pat. No. 7,147,855 whichclaims priority to U.S. Provisional Patent Application Nos. 60/338,287filed Dec. 7, 2001 and 60/382,004 filed May 20, 2002.

FIELD OF THE INVENTION

The present invention is in the general fields of molecular biology,cell biology, protein-protein interactions, autoimmunity, cancer, anddrug discovery.

BACKGROUND

Goodpasture antigen binding protein (GPBP) is a ubiquitous proteinkinase with a M_(r) of 80-89 kDa that is preferentially expressed intissues and cells that are common targets of autoimmune responses, suchas the Langerhans islets (type I diabetes); the white matter of thecentral nervous system (multiple sclerosis); the biliary ducts (primarybiliary cirrhosis); the cortical cells of the adrenal gland (Addisondisease); striated muscle cells (myasthenia gravis); spermatogonium(male infertility); Purkinje cells of the cerebellum (paraneoplasiccerebellar degeneration syndrome); and intestinal epithelial cells(pernicious anemia, autoimmune gastritis and enteritis).

GPBP is expressed as two isoforms (GPBP and GPBPΔ26) which result fromexon alternative splicing of the corresponding pre-mRNA. GPBP is themore active variant, and its expression is still more restricted tohistological structures targeted by common autoimmune responsesincluding human alveolar and glomerular basement membranes (Goodpasturedisease). GPBP binds to and phosphorylates the human α3 NC1 domain oftype IV collagen (α3(IV)NC1) also called the Goodpasture antigen (WO00/50607), as this domain is the target of the pathogenic autoantibodiesmediating the Goodpasture autoimmune response. Phosphorylation activatesthe α3(IV)NC1 domain for aggregation, a process that is catalyzed atleast in part by GPBP and which comprises conformational isomerizationreactions and disulfide-bond exchange (WO 02/061430).

An augmented expression of GPBP with respect to GPBPΔ26 has beenassociated with the production of non-tolerized, aberrant conformationalversions of the human α3(IV)NC1 domain (“aberrant conformers”) and thesubsequent autoantibody production that causes Goodpasture disease (WO02/061430). The evidence suggests that a similar pathogenic mechanism isinvolved in other autoimmune conditions, including cutaneous lupuserythematosus, pemphigus, pemphigoid and lichen planus, and thataberrant GPBP expression and autoimmune pathogenesis are relatedprocesses. Furthermore, GPBP is down-regulated in cancer cell lines (WO00/50607), suggesting that the cell machinery harboring GPBP/GPBPΔ26 isalso involved in signaling pathways that decrease cell division orinduce cell death. These pathways could be up regulated duringautoimmune pathogenesis to cause altered antigen presentation inindividuals carrying specific MHC haplotypes, and down regulated duringcell transformation to prevent autoimmune attack of the transformedcells during tumor growth.

Based on all of the above, there exists a need in the art to identifymethods and reagents for modifying GPBP activity for use in treatingautoimmune disorders and cancer.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides isolated GPBP-interacting90 and 130 kDa polypeptides, and portions thereof (GIP90/130polypeptides), antibodies to the GIP 90/130 polypeptides, andpharmaceutical compositions thereof. In a further aspect, the presentinvention provides isolated GIP90/130 nucleic acid sequences, expressionvectors comprising the nucleic acid sequences, and host cellstransfected with the expression vectors. The invention further providesmethods for detecting the GIP90/130 polypeptides or nucleic acidsequences, methods for modifying interactions between GPBP and GIP90/130polypeptides, aggregation of GIP90/130 polypeptides, and GIP90/130polypeptide-mediated gene transcription, and methods for treatingpatients with autoimmune disorders or cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the exon-intron structure of the GIP90 genomicDNA as determined by BLAST search against Human Genome NCBI in May 20,2002.

FIG. 2 is a representation of differences between various GIP90/130 mRNAand polypeptide species.

FIG. 3 is a sequence alignment of the full length GIP90/130 polypeptidesand DOC1 and DOC1-related protein.

FIG. 4 is the amino acid sequence of I-20. Residues in bold font arethose identified as essential for interactions between GIP90/130 andGPBP; in small letters are other residues identified as participating ininteraction between GIP90/130 and GPBP, but not essential; andunderlined are the residues implicated in GIP90/130 aggregation.

DETAILED DESCRIPTION OF THE INVENTION

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog(Ambion, Austin, Tex.).

As used herein, the term “GIP90/130” and “GIP90/130 polypeptide(s)”refers to the family of GPBP-interacting proteins that includes GIP90,GIP130a, GIP130b, and GIP130c, amino acid sequences derived therefrom,and includes both monomers and oligomers thereof.

As used herein, the term “GIP90” refers to the 90 kDa form of GIP, whichconsists of the amino acid sequence of SEQ ID NO:10, and includes bothmonomers and oligomers thereof.

As used herein, the term “GIP130a” refers to one of the 130 kDa forms ofGIP, which consists of the amino acid sequence of SEQ ID NO:12, andincludes both monomers and oligomers thereof.

As used herein, the term “GIP130b” refers to one of the 130 kDa forms ofGIP, which consists of the amino acid sequence of SEQ ID NO:14, andincludes both monomers and oligomers thereof.

As used herein, the term “GIP130c” refers to one of the 130 kDa forms ofGIP, which consists of the amino acid sequence of SEQ ID NO:16, andincludes both monomers and oligomers thereof.

The numbering of nucleotides and residues used below for GIP proteinsrefer to the GenBank accession number AF329092.

As used herein, the term “DOC proteins” or “DOC1 proteins” refers todown regulated in ovarian cancer-1 (DOC1) (Genbank accession number NM014890) and DOC1-related protein (Genbank accession number BC027860).DOC1 and DOC1-related protein are derived from the same gene since theyare identical in the homology region at nucleotide and amino acid levels

As used herein, the term “GPBP” refers to Goodpasture antigen bindingprotein, and includes both monomers and oligomers thereof, as disclosedin WO 00/50607.

As used herein, the term “GPBPΔ26” refers to the Goodpasture antigenbinding protein alternatively spliced product deleted for 26 amino acidresidues as disclosed in WO 00/50607, and includes both monomers andoligomers thereof.

As used herein pol κmeans the primary protein product of the POLK asdisclosed in WO 02/46378.

As used herein, pol κ76 means the 76 kDa alternatively spliced isoformproduct of the POLK as disclosed in WO 02/46378.

As used herein, “aggregation” refers to both self-aggregation of anindividual GIP90/130 polypeptide, and aggregation of two or moredifferent GIP90/130 polypeptides.

In one aspect, the present invention provides isolated GIP90/130polypeptides. In one embodiment, the isolated GIP90/130 polypeptidecomprises at least 6 amino acids of the amino acid sequence of SEQ IDNO:2, which is a unique 10 amino acid polypeptide (SYRRILGQLL) that isherein demonstrated to be essential for the interaction betweenGIP90/130 and GPBP (discussed in detail below), and is not present inDOC proteins. In further embodiments, the isolated GIP90/130 polypeptidecomprises at least 7, 8, 9, or 10 amino acids of the amino acid sequenceof SEQ ID NO:2. In still further embodiments, the isolated GIP90/130polypeptide consists of at least 6, 7, 8, 9, or 10 amino acids of theamino acid sequence of SEQ ID NO:2. These polypeptides can be used, forexample, to modify interactions between GPBP and GIP90/130 polypeptidesor to raise antibodies that interfere with GPBP-GIP90/130 interaction.

In further embodiments, the isolated GIP90/130 polypeptide comprisesand/or consists of the amino acid sequence of SEQ ID NO:4, which is theN-terminal region of GIP90/130a/c that is not present in DOC proteins(described in detail below), and which is encoded by exon II-IV and partof exon V (FIG. 3). These polypeptides are thus useful, for example, todevelop reagents, such as antibodies, that can distinguish betweenGIP90/130 and DOC proteins. This polypeptide includes sequencesimplicated in the interaction between GPBP and GIP90/130 (including SEQID NO:2), and thus can be used (or antibodies to the polypeptides can beused), for example, to modify interactions between GPBP and GIP90/130polypeptides. This polypeptide also includes sequences implicated inGIP90/130 aggregation, and thus can further be used (or antibodies tothe polypeptides can be used) to modify GIP90/130 aggregation. Thispolypeptide also includes sequences implicated in the transcriptionalactivity of GIP90/130 and thus the polypeptides, or antibodies derivedtherefrom, can be further used for modulating specific gene expression.

The polypeptides of the invention also include polypeptides comprisingand/or consisting of the amino acid sequence of SEQ ID NO:6, which isreferred to as I-20, a 265 amino acid polypeptide that is described indetail below. This polypeptide interacts more strongly with GPBP and polκ76 than the full length GIP90/130 polypeptides, and aggregates moreefficiently than the full length GIP90/130 polypeptides. Furthermore,I-20 does not induce gene transcription, in contrast to the full lengthGIP90/130 polypeptides. Therefore this polypeptide can be used (orantibodies to the polypeptides can be used), for example, to modify (a)interactions between GPBP and GIP90/130 polypeptides; (b) interactionsbetween pol κ76 and GIP90/130 polypeptides; (c) GIP90/130 polypeptideaggregation; and (d) other functions of the GIP90/130 polypeptides, suchas induction of gene transcription.

The polypeptides of the invention also include polypeptides comprisingand/or consisting of the amino acid sequence of SEQ ID NO:8, whichconsists of the N-terminus of GIP90 to the end of I-20, and is encodedby exons II-IV and part of exon V up to the end of the I-20 codingsequence. This polypeptide includes sequences implicated in (a) theinteraction between GPBP and GIP90/130 polypeptides, (b) GIP90/130polypeptide aggregation, and (c) the transcriptional activity ofGIP90/130 polypeptides, and thus the polypeptides, or antibodies derivedtherefrom, can be used, for example, to modify interactions between GPBPand GIP90/130 polypeptides, to modify GIP90/130 aggregation, and tomodulate gene expression.

The polypeptides of the invention also include polypeptides comprisingand/or consisting of the amino acid sequence of SEQ ID NO:10 (GIP90),SEQ ID NO:12 (GIP130a), SEQ ID NO:14 (GIP130b), or SEQ ID NO:16(GIP130c). These full length polypeptides, described in more detailbelow, interact with GPBP and are capable of aggregation. Thesepolypeptides can be used, for example, to modify GPBP-GIP90/130interactions, to modify GIP90/130 aggregation, to modulate geneexpression, as well as for other purposes described herein.

In a further embodiment, the isolated GIP 90/130 polypeptide comprisesat least 8 amino acids of the amino acid sequence of SEQ ID NO:18, whichis a unique 15 amino acid peptide that is present at the C-terminus ofGIP90 and is not present in DOC proteins, GIP130a, GIP130b, or GIP130c,and thus can be used, for example, to generate reagents, such asantibodies, to distinguish GIP90 from other members of the GIP90/130polypeptide family. Furthermore, the polypeptides, or antibodiesthereto, can be used to specifically modify GIP90 self-aggregation. Infurther embodiments, the isolated GIP90/130 polypeptide comprises orconsists of at least 9, 10, 11, 12, 13, 14, or 15 amino acids of theamino acid sequence of SEQ ID NO:18.

In a further embodiment, the isolated GIP90/130 polypeptide consists ofat least 8 amino acids of the amino acid sequence of SEQ ID NO:20, whichis a 30 amino acid polypeptide present within I-20 that has beenimplicated in the interaction of GIP90/130 with GPBP and also inGIP90/130 aggregation. In further embodiments, the isolated GIP90/130polypeptide consists of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids the aminoacid sequence of SEQ ID NO:20. Thus, these polypeptides, or antibodiesto the polypeptides, can be used, for example, to modify interactionsbetween GPBP and GIP90/130 polypeptides. Furthermore, since thispolypeptide is present in each of GIP90, GIP130a, GIP130b, GIP130c, andDOC1 proteins, these polypeptides, or antibodies thereto, can be used togenerally modify aggregation of the GIP90/130 polypeptides and DOC1proteins. Despite the fact that DOC1 proteins contain SEQ ID NO:20, theydo not interact in a two hybrid assay with GPBP (see below), and thusSEQ ID NO:20, while implicated in the interaction of GIP90/130polypeptides and GPBP, is not sufficient for GPBP interaction.

In a still further embodiment, the isolated GIP90/130 polypeptidecomprises or consists of the amino acid sequence of SEQ ID NO:22, whichis a unique 386 amino acid polypeptide that is present at the C-terminusof GIP130a but is not present in GIP90, is not wholly present in DOC1,and includes variations from GIP130b, GIP130c, and DOC1-related protein,and thus can be used, for example, to modify GIP130a aggregation, and togenerate reagents, such as antibodies, to distinguish GIP130a from othermembers of the GIP90/130 polypeptide family, and the DOC proteins. Thisregion contains sequences that down-regulate GIP90/130 interaction withGPBP which can be used to modify GIP90/130-GPBP interaction, or togenerate reagents, such as antibodies for the same purposes.

In a still further embodiment, the isolated GIP90/130 polypeptidecomprises or consists of the amino acid sequence of SEQ ID NO:24, whichis GIP130a deleted from the N-terminus to the end of I-20. Thispolypeptide lacks critical regions of the GIP90/130 polypeptidesimplicated in GPBP interaction and induction of gene expression, andlike the C terminus of GIP130b/c contains amino acid sequences thatdown-regulate interaction with GPPB. Thus, the polypeptides, orantibodies thereto, can be used, for example, to modify GPBP-GIP90/130polypeptide interactions or to modify GIP90/130 polypeptide aggregation.

In a still further embodiment, the isolated GIP90/130 polypeptidecomprises or consists of the amino acid sequence of SEQ ID NO:26, whichis a unique 7 amino acid polypeptide present at the C-terminus ofGIP130a, and is not present in any of GIP90, GIP130b, GIP130c, and DOCproteins. Thus, these polypeptides can be used to produce reagents, suchas antibodies, that are specific for GIP130a, and which can be used, forexample, to specifically modify GIP130a aggregation.

In another embodiment, the isolated GIP90/130 polypeptide comprises atleast 6 amino acids of the amino acid sequence of SEQ ID NO:28, which isa unique 10 amino acid polypeptide (LDKVVEKHKE) within I-20 thatparticipates in interactions between GIP90/130 polypeptides and GPBP, isessential for GIP90/130 polypeptide aggregation, and is not present inDOC proteins. In further embodiments, the isolated GIP90/130 polypeptidecomprises or consists of at least 7, 8, 9, or 10 amino acids of theamino acid sequence of SEQ ID NO:28. These polypeptides or antibodiesraised against them can be used, for example, to modify interactionsbetween GPBP and GIP90/130 polypeptides or to modify GIP90/130polypeptide aggregation.

In another embodiment, the isolated GIP90/130 polypeptide consists of atleast 6 amino acids of the amino acid sequence of SEQ ID NO:30, which isan 10 amino acid polypeptide (EEEQKATRLE) within I-20 that participatesin interactions between GIP90/130 polypeptides and GPBP, is essentialfor GIP90/130 polypeptide aggregation, and is present in DOC proteins.In further embodiments, the isolated GIP90/130 polypeptide consists ofat least 7, 8, 9, or 10 amino acids of the amino acid sequence of SEQ IDNO:30. These polypeptides or antibodies raised against them can be used,for example, to modify interactions between GPBP and GIP90/130polypeptides or to modify GIP90/130 polypeptide aggregation.Furthermore, since this polypeptide is present in each of GIP90,GIP130a, GIP130b, GIP130c, and DOC1 proteins, these polypeptides, orantibodies thereto, can be used to generally modify aggregation of theGIP90/130 polypeptides and DOC1/DOC1-related proteins. Despite the factthat DOC1 proteins contain SEQ ID NO:20, they do not interact in a twohybrid assay with GPBP (see below), and thus SEQ ID NO:20, whileimplicated in the interaction of GIP90/130 polypeptides and GPBP, is notsufficient for GPBP interaction.

In another embodiment, the isolated GIP90/130 polypeptide comprises atleast 8 amino acids of the amino acid sequence of SEQ ID NO:32, which isa unique 20 amino acid polypeptide (LDKVVEKHKESYRRILGQLL) within I-20that contains essential residues for the interaction between GIP90/130polypeptides and GPBP and for GIP90/130 polypeptide aggregation, and isnot present in DOC proteins. In further embodiments, the isolatedGIP90/130 polypeptide comprises or consists of at least 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 amino acids of the amino acid sequenceof SEQ ID NO:32. These polypeptides can be used, for example, to modifyinteractions between GPBP and GIP90/130 polypeptides and to modifyGIP90/130 polypeptide aggregation, or to raise antibodies that modifyinteractions between GPBP and GIP90/130 polypeptides and to modifyGIP90/130 polypeptide aggregation.

In another embodiment, the isolated GIP90/130 polypeptide consists of atleast 8 amino acids of the amino acid sequence of SEQ ID NO:34, which isa 50 amino acid polypeptide that is contained within I-20, containsregions essential for the interaction between GIP90/130 polypeptides andGPBP and for GIP90/130 polypeptide aggregation, and is present in DOCproteins. In further embodiments, the isolated GIP90/130 polypeptideconsists of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids of the aminoacid sequence of SEQ ID NO:34. These polypeptides can be used, forexample, to modify interactions between GPBP and GIP90/130 polypeptidesand to modify GIP90/130 polypeptide aggregation, or to raise antibodiesthat modify interactions between GPBP and GIP90/130 polypeptides and tomodify GIP90/130 polypeptide aggregation. Furthermore, since thispolypeptide is present in each of GIP90, GIP130a, GIP130b, GIP130c, andDOC1 proteins, these polypeptides, or antibodies thereto, can be used togenerally modify aggregation of the GIP90/130 polypeptides andDOC1/DOC1-related proteins. Despite the fact that DOC1 proteins containSEQ ID NO:20, they do not interact in a two hybrid assay with GPBP (seebelow), and thus SEQ ID NO:20, while implicated in the interaction ofGIP90/130 polypeptides and GPBP, is not sufficient for GPBP interaction.

The polypeptides of the invention also include polypeptides comprisingand/or consisting of the amino acid sequence of SEQ ID NO:36, whichconsists of the first 240 amino acids of the N-terminus of GIP130b,which is not present in DOC1 proteins, and which differs from thecorresponding sequence in GIP90, GIP130a, and GIP130c by a single aminoacid residue at position 168. This polypeptide includes sequencesimplicated in (a) the interaction between GPBP and GIP90/130polypeptides, (b) GIP90/130 polypeptide aggregation, and (c) thetranscriptional activity of GIP90/130 polypeptides, and thus thepolypeptides, or antibodies derived therefrom, can be used, for example,to modify interactions between GPBP and GIP90/130 polypeptides, tomodify GIP90/130 aggregation, and to modulate gene expression.

In a still further embodiment, the isolated GIP90/130 polypeptideconsists of the amino acid sequence of SEQ ID NO:38 which is a unique384 amino acid polypeptide that is present at the C terminus ofGIP130b/c and DOC1-related protein but is not present in GIP90, is notwholly present in DOC1, and includes variations from GIP130a, and thuscan be used, for example, to modify GIP30b/c aggregation, and togenerate reagents, such as antibodies, to distinguish GIP130 b/c and theDOC1-related protein from other members of the GIP90/130 polypeptidefamily.

As used herein, an “isolated polypeptide” refers to a polypeptide thatis substantially free of other proteins, cellular material and culturemedium when isolated from cells or produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. Thus, the protein can either be purified from naturalsources, chemically synthesized, or recombinant protein can be purifiedfrom the recombinant host cells disclosed below.

Synthetic polypeptides, prepared using the well known techniques ofsolid phase, liquid phase, or peptide condensation techniques, or anycombination thereof, can include natural and unnatural amino acids.Amino acids used for peptide synthesis may be standard Boc (Nα-aminoprotected Nα-t-butyloxycarbonyl) amino acid resin with the standarddeprotecting, neutralization, coupling and wash protocols of theoriginal solid phase procedure of Merrifield (1963, J. Am. Chem. Soc.85:2149-2154), or the base-labile Nα-amino protected9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpinoand Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and Boc Nα-aminoprotected amino acids can be obtained from Sigma, Cambridge ResearchBiochemical, or other chemical companies familiar to those skilled inthe art. In addition, the polypeptides can be synthesized with otherNα-protecting groups that are familiar to those skilled in this art.

Solid phase peptide synthesis may be accomplished by techniques familiarto those in the art and provided, for example, in Stewart and Young,1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co.,Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res.35:161-214, or using automated synthesizers. The polypeptides of theinvention may comprise D-amino acids (which are resistant to L-aminoacid-specific proteases in vivo), a combination of D- and L-amino acids,and various “designer” amino acids (e.g., β-methyl amino acids,Cα-methyl amino acids, and Nα-methyl amino acids, etc.) to conveyspecial properties. Synthetic amino acids include ornithine for lysine,fluorophenylalanine for phenylalanine, and norleucine for leucine orisoleucine.

In addition, the polypeptides can have peptidomimetic bonds, such asester bonds, to prepare peptides with novel properties. For example, apeptide may be generated that incorporates a reduced peptide bond, i.e.,R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues or sequences. Areduced peptide bond may be introduced as a dipeptide subunit. Such apolypeptide would be resistant to protease activity, and would possessan extended half-live in vivo.

Alternatively, the proteins are produced by the recombinant host cellsdisclosed below, and purified using standard techniques. (See forexample, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press.)) The protein can thus be purifiedfrom prokaryotic or eukaryotic sources. In various further preferredembodiments, the protein is purified from bacterial, yeast, or mammaliancells.

The protein may comprise additional sequences useful for promotingpurification of the protein, such as epitope tags and transport signals.Examples of such epitope tags include, but are not limited to FLAG(Sigma Chemical, St. Louis, Mo.), myc (9E10) (Invitrogen, Carlsbad,Calif.), 6-His (Invitrogen; Novagen, Madison, Wis.), and HA (BoehringerManheim Biochemicals). Examples of such transport signals include, butare not limited to, export signals, secretory signals, nuclearlocalization signals, and plasma membrane localization signals.

In another aspect, the present invention provides antibodies against theGIP90/130 polypeptides disclosed herein. Such antibodies can be used ina manner similar to the polypeptides they recognize in modifyingGPBP-GIP90/130 interactions, modifying GIP90/130 aggregation, and/ormodifying GIP90/130-mediated transcriptional activity. Furthermore, suchantibodies can be used to distinguish between members of the GIP90/130family, as discussed above.

In one embodiment, the antibodies are directed against an epitopepresent in a polypeptide of one or more of the amino acid sequencesselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:18, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, and SEQ ID NO:36. In afurther embodiment, the antibodies are directed against an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, and SEQ ID NO:38.

Antibodies can be made by well-known methods, such as described inHarlow and Lane, Antibodies; A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988). In one example, pre-immuneserum is collected prior to the first immunization. A peptide portion ofthe amino acid sequence of a GIP90/130 polypeptide, together with anappropriate adjuvant, is injected into an animal in an amount and atintervals sufficient to elicit an immune response. Animals are bled atregular intervals, preferably weekly, to determine antibody titer. Theanimals may or may not receive booster injections following the initialimmunization. At about 7 days after each booster immunization, or aboutweekly after a single immunization, the animals are bled, the serumcollected, and aliquots are stored at about −20° C. Polyclonalantibodies against GIP90/130 polypeptides can then be purified directlyby passing serum collected from the animal through a column to whichnon-antigen-related proteins prepared from the same expression systemwithout GIP90/130 polypeptides bound.

Monoclonal antibodies can be produced by obtaining spleen cells from theanimal. (See Kohler and Milstein, Nature 256, 495-497 (1975)). In oneexample, monoclonal antibodies (mAb) of interest are prepared byimmunizing inbred mice with a GIP90/130 polypeptide, or portion thereof.The mice are immunized by the IP or SC route in an amount and atintervals sufficient to elicit an immune response. The mice receive aninitial immunization on day 0 and are rested for about 3 to about 30weeks. Immunized mice are given one or more booster immunizations of bythe intravenous (IV) route. Lymphocytes from antibody positive mice areobtained by removing spleens from immunized mice by standard proceduresknown in the art. Hybridoma cells are produced by mixing the spleniclymphocytes with an appropriate fusion partner under conditions whichwill allow the formation of stable hybridomas. The antibody producingcells and fusion partner cells are fused in polyethylene glycol atconcentrations from about 30% to about 50%. Fused hybridoma cells areselected by growth in hypoxanthine, thymidine and aminopterinsupplemented Dulbecco's Modified Eagles Medium (DMEM) by proceduresknown in the art. Supernatant fluids are collected from growth positivewells and are screened for antibody production by an immunoassay such assolid phase immunoradioassay. Hybridoma cells from antibody positivewells are cloned by a technique such as the soft agar technique ofMacPherson, Soft Agar Techniques, in Tissue Culture Methods andApplications, Kruse and Paterson, Eds., Academic Press, 1973.

To generate such an antibody response, a GIP90/130 polypeptide orportion thereof is typically formulated with a pharmaceuticallyacceptable carrier for parenteral administration. Such acceptableadjuvants include, but are not limited to, Freund's complete, Freund'sincomplete, alum-precipitate, water in oil emulsion containingCorynebacterium parvum and tRNA. The formulation of such compositions,including the concentration of the polypeptide and the selection of thevehicle and other components, is within the skill of the art.

The term antibody as used herein is intended to include antibodyfragments thereof which are selectively reactive with GIP90/130polypeptides. Antibodies can be fragmented using conventionaltechniques, and the fragments screened for utility in the same manner asdescribed above for whole antibodies. For example, F(ab′)₂ fragments canbe generated by treating antibody with pepsin. The resulting F(ab′)₂fragment can be treated to reduce disulfide bridges to produce Fab′fragments.

In another aspect, the present invention provides isolated nucleic acidsthat encode GIP90/130 polypeptides. In one embodiment, the isolatednucleic acid sequences comprise sequences encoding an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:32, and SEQ ID NO:36. In a further embodiment, theisolated nucleic acid sequences consist of sequences encoding an aminoacid sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO;34, SEQ ID NO:36, and SEQ ID NO:38.

In another embodiment, the isolated nucleic acids comprise sequencesthat hybridize under high stringency conditions to a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, and SEQ IDNO:35, their complement, or their transcription product. Stringency ofhybridization is used herein to refer to conditions under which nucleicacid hybrids are stable. As known to those of skill in the art, thestability of hybrids is reflected in the melting temperature (T_(M)) ofthe hybrids. T_(M) decreases approximately 1-1.5° C. with every 1%decrease in sequence homology. In general, the stability of a hybrid isa function of sodium ion concentration and temperature. Typically, thehybridization reaction is performed under conditions of lowerstringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions. Thus, as used herein, high stringency refers to conditionsthat permit hybridization of those nucleic acid sequences that formstable hybrids in 0.1% SSPE at 65° C. It is understood that theseconditions may be duplicated using a variety of buffers and temperaturesand that they are not necessarily precise. Denhardt's solution and SSPE(see, e.g., Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989) are wellknown to those of skill in the art, as are other suitable hybridizationbuffers.

In another embodiment, the isolated nucleic acids comprise one or moresequences selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, and SEQ IDNO:35, their complement, or their transcription product. In a furtherembodiment, the isolated nucleic acid sequences comprise one or moresequences selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:31, and SEQ ID NO:35, their complement, or theirtranscription product. In a further embodiment, the isolated nucleicacid sequences consist of one or more sequences selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37, theircomplement, or their transcription product.

As used herein, an “isolated nucleic acid sequence” refers to a nucleicacid sequence that is free of gene sequences which naturally flank thenucleic acid in the genomic DNA of the organism from which the nucleicacid is derived (i.e., genetic sequences that are located adjacent tothe gene for the isolated nucleic molecule in the genomic DNA of theorganism from which the nucleic acid is derived). An “isolated”GIP90/130 nucleic acid sequence according to the present invention may,however, be linked to other nucleotide sequences that do not normallyflank the recited sequence, such as a heterologous promoter sequence, orother vector sequences. It is not necessary for the isolated nucleicacid sequence to be free of other cellular material to be considered“isolated”, as a nucleic acid sequence according to the invention may bepart of an expression vector that is used to transfect host cells (seebelow).

In all of these embodiments, the isolated nucleic acid sequence maycomprise RNA or DNA, and may be single stranded or double stranded. Suchsingle stranded sequences can comprise the disclosed sequence, itscomplement, or the transcription product thereof. The isolated sequencemay further comprise additional sequences useful for promotingexpression and/or purification of the encoded protein, including but notlimited to polyA sequences, modified Kozak sequences, and sequencesencoding epitope tags, export signals, and secretory signals, nuclearlocalization signals, and plasma membrane localization signals.

In another embodiment, the present invention provides an expressionvector comprising an isolated nucleic acid as described above,operatively linked to a promoter. In a preferred embodiment, thepromoter is heterologous (i.e.: is not the naturally occurring GIP90/130promoter). A promoter and a GIP90/130 nucleic acid sequence are“operatively linked” when the promoter is capable of driving expressionof the GIP90/130 DNA into RNA.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA into which additional DNA segments may be cloned.Another type of vector is a viral vector, wherein additional DNAsegments may be cloned into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors), are integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of nucleic acid sequences to whichthey are operatively linked. Such vectors are referred to herein as“recombinant expression vectors” or simply “expression vectors”. In thepresent invention, the expression of any nucleic acid sequence isdirected by operatively linking the promoter sequences of the inventionto the nucleic acid sequence to be expressed. In general, expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids. In the present specification, “plasmid” and “vector” may beused interchangeably as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions.

The vector may also contain additional sequences, such as a polylinkerfor subcloning of additional nucleic acid sequences and apolyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed, including but not limited to the SV40 andbovine growth hormone poly-A sites. The vector may further include atermination sequence, which can serve to enhance message levels and tominimize read through from the construct into other sequences. Finally,expression vectors typically have selectable markers, often in the formof antibiotic resistance genes, that permit selection of cells thatcarry these vectors.

In a further embodiment, the present invention provides recombinant hostcells in which the expression vectors disclosed herein have beenintroduced. As used herein, the term “host cell” is intended to refer toa cell into which a nucleic acid of the invention, such as a recombinantexpression vector of the invention, has been introduced. Such cells maybe prokaryotic or eukaryotic.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It should be understood that such terms refernot only to the particular subject cell but to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

The host cells can be transiently or stably transfected with one or moreof the expression vectors of the invention. Such transfection ofexpression vectors into prokaryotic and eukaryotic cells can beaccomplished via any technique known in the art, including but notlimited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome mediated-, DEAE dextranmediated-, polycationic mediated-, or viral mediated transfection.Alternatively, the host cells can be infected with a recombinant viralvector comprising the GIP90/130 nucleic acid. (See, for example,Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press; Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N.Y.).

In a further aspect, the invention provides methods for detecting thepresence of the GIP90/130 polypeptides in a protein sample, comprisingproviding a protein sample to be screened, contacting the protein sampleto be screened with an antibody against one or more GIP90/130polypeptides, and detecting the formation of antibody-GIP90/130polypeptide complexes. The antibody can be either polyclonal ormonoclonal, although monoclonal antibodies are preferred. As usedherein, the term “protein sample” refers to any sample that may containGIP90/130 polypeptides, including but not limited to tissues andportions thereof, tissue sections, intact cells, cell extracts, purifiedor partially purified protein samples, bodily fluids, and nucleic acidexpression libraries. Accordingly, this aspect of the present inventionmay be used to test for the presence of GIP90/130 polypeptides in thesevarious protein samples by standard techniques including, but notlimited to, immunolocalization, immunofluorescence analysis, Westernblot analysis, ELISAs, and nucleic acid expression library screening,(See for example, Sambrook et al, 1989.) In one embodiment, thetechniques may determine only the presence or absence of GIP90/130polypeptides. Alternatively, the techniques may be quantitative, andprovide information about the relative amount of GIP90/130 polypeptidesin the sample. For quantitative purposes, ELISAs are preferred.

Detection of immunocomplex formation between GIP90/130 polypeptides andantibodies or fragments thereof directed against GIP90/130 polypeptidescan be accomplished by standard detection techniques. For example,detection of immunocomplexes can be accomplished by using labeledantibodies or secondary antibodies. Such methods, including the choiceof label are known to those ordinarily skilled in the art. (Harlow andLane, Supra). Alternatively, the polyclonal or monoclonal antibodies canbe coupled to a detectable substance. The term “coupled” is used to meanthat the detectable substance is physically linked to the antibody.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examplesof suitable prosthetic-group complexes include streptavidin/biotin andavidin/biotin. Examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anexample of a luminescent material includes luminol. Examples of suitableradioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Such methods of detection are useful for a variety of purposes,including but not limited to detecting an autoimmune condition,identifying cell division arrest or cell death, detecting GIP90/130interactions with GPBP or other proteins, immunolocalization ofGIP90/130 polypeptides in a tissue sample, Western blot analysis, andscreening of expression libraries to find related proteins.

In yet another aspect, the invention provides methods for detecting thepresence of nucleic acid sequences encoding GIP90/130 polypeptides in asample comprising providing a nucleic acid sample to be screened,contacting the sample with a nucleic acid probe derived from theisolated nucleic acid sequences of the invention, or fragments thereof,and detecting complex formation.

As used herein, the term “sample” refers to any sample that may containa GIP90/130 polypeptide-encoding nucleic acid, including but not limitedto tissues and portions thereof, tissue sections, intact cells, cellextracts, purified or partially purified nucleic acid samples, DNAlibraries, and bodily fluids. Accordingly, this aspect of the presentinvention may be used to test for the presence of GIP90/130polypeptide-encoding mRNA or DNA in these various samples by standardtechniques including, but not limited to, in situ hybridization,Northern blotting, Southern blotting, DNA library screening, polymerasechain reaction (PCR) or reverse transcription-PCR (RT-PCR). (See forexample, Sambrook et al, 1989.) In one embodiment, the techniques maydetermine only the presence or absence of the nucleic acid of interest.Alternatively, the techniques may be quantitative, and provideinformation about the relative amount of the nucleic acid of interest inthe sample. For quantitative purposes, quantitative PCR and RT-PCR arepreferred. Thus, in one example, RNA is isolated from a sample, andcontacted with an oligonucleotide derived from the GIP90/130polypeptide-encoding nucleic acid sequence, together with reversetranscriptase, under suitable buffer and temperature conditions toproduce cDNAs from the GIP90/130 RNA. The cDNA is then subjected to PCRusing primer pairs derived from the appropriate nucleic acid sequencedisclosed herein. In a preferred embodiment, the primers are designed todetect the presence of the RNA expression product of GIP90/130, and theamount of GIP90/130 gene expression in the sample is compared to thelevel in a control sample.

For detecting GIP90/130 nucleic acid sequences, standard labelingtechniques can be used to label the probe, the nucleic acid of interest,or the complex between the probe and the nucleic acid of interest,including, but not limited to radio-, enzyme-, chemiluminescent-, oravidin or biotin-labeling techniques, all of which are well known in theart. (See, for example, Molecular Cloning: A Laboratory Manual(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), GeneExpression Technology (Methods in Enzymology, Vol. 185, edited by D.Goeddel, 1991. Academic Press, San Diego, Calif.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.)).

Such methods of nucleic acid detection are useful for a variety ofpurposes, including but not limited to detecting an autoimmunecondition, identifying cell division arrest or cell death, identifyingcells that express GIP90/130 nucleic acid sequences, in situhybridization for GIP90/130 gene expression, Northern and Southern blotanalysis, and DNA library screening.

As discussed above, GIP90/130 polypeptides are likely to be involved incell signaling pathways that impair cell division or cause cell death,which are thought to be up-regulated during autoimmune pathogenesis anddown-regulated in cancer cells to prevent autoimmune attack during tumorgrowth. Thus, the detection methods disclosed herein can be used todetect cells that are undergoing such cell death-related processes.

Furthermore, the present invention provides method for treating anautoimmune disorder or cancer comprising modifying the expression oractivity of GIP90/130 RNA or GIP90/130 polypeptides, such as byincreasing or decreasing their expression or activity. Modifying theexpression or activity of GIP90/130 RNA or GIP90/130 polypeptides can beaccomplished by using specific inducers or inhibitors of GIP90/130polypeptide expression or activity, such as GIP90/130 antibodies,polypeptides representing interactive motifs of GIP90/130 such as thosedisclosed herein, antisense or RNA interference therapy based on thedesign of antisense oligonucleotides or double stranded RNAs to theGIP90/130 nucleic acid sequences disclosed herein, cell therapy usinghost cells expressing one or more GIP90/130 polypeptides, or othertechniques known in the art. As used herein, “modification of expressionor activity” refers to modifying expression or activity of either theRNA or protein product.

For example, knowing that the GIP90/130 gene is a tumor suppressor gene,that aberrantly increased cell death processes are the basis of specificautoimmune pathogenesis (WO 00/50607), and that aggregates of GIP90/130polypeptides are expressed in a number of human tissues that are commontarget of autoimmune responses, the administration of GIP90/130polypeptides or nucleic acids of the invention, particularly thoserepresenting essential interactive motifs for GIP90/130 polypeptideaggregation and/or interaction with other cellular components, such asGPBP, would impact pathogenesis and therefore serve as therapeuticagents for autoimmunity. Alternatively, tumor cells express little or noGPBP or GIP90/130, and thus the administration of the GIP90/130polypeptide or nucleic acid sequences of the invention, particularly thefull length GIP90, GIP130a, GIP130b, and/or GIP130c, alone or incombination with GPBP, is expected to provide a therapeutic benefit inpatients with cancer.

While not being limited to any specific mechanism of action, it isbelieved that a therapeutic benefit in cancer patients would be derivedby promoting GIP90/130 interactions with other cellular constituents,such as GPBP and/or GIP90/130 aggregation, whereas a therapeutic benefitto autoimmunity patients would be derived by inhibiting theseinteractions and/or aggregation.

In another aspect, the invention provides methods for modifyingGIP90/130 activity comprising contacting cells with an amount effectiveof one or more of the polypeptides, antibodies, nucleic acids, orpharmaceutical compositions thereof, of the invention to modifyGIP90/130 activity. Such cell contacting can be in vitro or in vivo, and“modifying” includes both increasing or decreasing GIP90/130 activity,including transcription-promoting activity.

In another aspect, the invention provides methods for modifying GPBPactivity, comprising contacting cells with an amount effective of one ormore of the polypeptides, antibodies, nucleic acids, or pharmaceuticalcompositions thereof, of the invention to modify GPBP activity. Suchcell contacting can be in vitro or in vivo, and “modifying” includesboth increasing or decreasing GPBP activity. For example, augmented GPBPactivity is associated with autoimmunity, and thus the administration ofthe GIP90/130 polypeptides or antibodies of the invention (or genetherapy by administration of the GIP90/130 nucleic acid sequences orvectors thereof of the invention) would be expected to impactGPBP-GIP90/130 interactions, and to provide a therapeutic benefit inpatients with an autoimmune disorder. Alternatively, tumor cells expresslittle or no GPBP, and thus the co-administration of the GIP90/130polypeptides of the invention, particularly the full length GIP90,GIP130a, GIP130b, and/or GIP130c, in combination with GPBP, would beexpected to provide a therapeutic benefit in patients with cancer.

In another aspect, the present invention provides methods for modifyingpol κ76 polypeptide activity, comprising contacting cells with an amounteffective of one or more of the polypeptides, antibodies, nucleic acids,or pharmaceutical compositions thereof, of the invention to modify polκ76 activity. Such cell contacting can be in vitro or in vivo, and“modifying” includes both increasing or decreasing pol κ76 activity. Forexample, augmented pol κ76 activity is associated with autoimmunity (WO02/46378), and thus the administration of the GIP90/130 polypeptides orantibodies of the invention (or gene therapy by administration of theGIP90/130 nucleic acid sequences or vectors thereof of the invention)would be expected to impact pol κ76-GIP90/130 interactions, and toprovide a therapeutic benefit in patients with an autoimmune disorder.

In practicing the therapeutic methods of the invention, the amount ordosage range of the GIP90/130 polypeptides or antibodies theretogenerally ranges between about 0.01 μg/kg body weight and about 10 mg/kgbody weight, preferably ranging between about 0.10 μg/kg and about 5mg/kg body weight, and more preferably between about 1 μg/kg and about 5mg/kg body weight.

In a further aspect, the present invention provides pharmaceuticalcompositions, comprising an amount effective of the GIP90/130polypeptides, antibodies thereto, and nucleic acids disclosed herein tocarry out one or more of the therapeutic methods of the invention, and apharmaceutically acceptable carrier. The GIP90/130 polypeptides, orantibodies thereto, may be subjected to conventional pharmaceuticaloperations such as sterilization and/or may contain conventionaladjuvants, such as preservatives, stabilizers, wetting agents,emulsifiers, buffers etc.

For administration, the polypeptides are ordinarily combined with one ormore adjuvants appropriate for the indicated route of administration.The compounds may be admixed with lactose, sucrose, starch powder,cellulose esters of alkanoic acids, stearic acid, talc, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine,and/or polyvinyl alcohol, and tableted or encapsulated for conventionaladministration. Alternatively, the compounds of this invention may bedissolved in saline, water, polyethylene glycol, propylene glycol,carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanutoil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.Other adjuvants and modes of administration are well known in thepharmaceutical art. The carrier or diluent may include time delaymaterial, such as glyceryl monostearate or glyceryl distearate alone orwith a wax, or other materials well known in the art.

The polypeptides or pharmaceutical compositions thereof may beadministered by any suitable route, including orally, parentally, byinhalation spray, rectally, or topically in dosage unit formulationscontaining conventional pharmaceutically acceptable carriers, adjuvants,and vehicles. The term parenteral as used herein includes, subcutaneous,intravenous, intra-arterial, intramuscular, intrasternal,intratendinous, intraspinal, intracranial, intrathoracic, infusiontechniques or intraperitoneally. In preferred embodiments, thepolypeptides are administered intravenously or subcutaneously.

The polypeptides may be made up in a solid form (including granules,powders or suppositories) or in a liquid form (e.g., solutions,suspensions, or emulsions). The polypeptides of the invention may beapplied in a variety of solutions. Suitable solutions for use inaccordance with the invention are sterile, dissolve sufficient amountsof the polypeptides, and are not harmful for the proposed application.

The present invention may be better understood with reference to theaccompanying examples that are intended for purposes of illustrationonly and should not be construed to limit the scope of the invention, asdefined by the claims appended hereto.

EXAMPLES

Identification and Characterization of GIP90/130 Polypeptides

We performed a yeast two-hybrid screening on several human cDNAlibraries searching for GPBP-interactive proteins. The screenings wereperformed using full length GPBP as bait, cloned in vector pGBT9 togenerate the GAL4 binding domain-fusion protein. With the resultingconstruct we transformed yeast HF7c cells to obtain a stably transfectedcell line which was subsequently transformed with the different cDNAlibraries we have used: Human Skeletal Muscle (pGAD10 vector), HumanKidney (pGAD10), Human Pancreas (pGAD10), Human Brain (pACT2) and Hela(pGADGH) cDNA libraries (all from Clontech). The transformations werecarried out according to the supplier's instructions and plated onmedium deficient in Trp, Leu and His containing 20 mM3-amino-1,2,4-triazol. Interactions were assessed following themanufacture's recommendations. Specifically β-galactosidase activity wasassayed with X-GAL (0.75 mg/ml) for the lift colony assays and withortho-nitrophenyl β-D galactopyranoside (0.66 mg/ml) for the in-solutiondeterminations.

We isolated an 800 bp cDNA (“I-20 cDNA”) encompassing an open readingframe (ORF) which encodes a 265 residue polypeptide, I-20 (SEQ ID NO:6);from a human skeletal muscle library. Part of the ORF coincided with theORF encoding DOC1 (down-regulated in ovarian cancer 1) (GenBankaccession NP_(—)055705) (Mok et al., Gynecol. Oncol. 52(2):247-252(1994)), a polypeptide whose encoding mRNA is not found in ovariancancer cell lines, but is abundantly expressed in normal ovarian celllines. For this reason, the DOC-1 gene is considered to be a tumorsuppressor gene.

Using the I-20 cDNA, we probed a multi-tissue Northern blot (Clontech)to determine the level of expression of the I-20 encoding mRNA in normalhuman tissues and in a number of human cancer cell lines. The membraneswere hybridized with ³²P-α-dCTP labelled I-20 cDNA (SEQ ID NO:5), andspecific mRNAs species were identified by autoradiography. We identifiedfour MRNA species of 9, 4.4, 4 and 3 Kb. The species of 9, 4.4 and 3 Kbwere more abundant in skeletal muscle, while the 4 Kb species displayedsimilar expression in skeletal muscle, pancreas and lung, and higherexpression in heart tissue. With the exception of heart, which containedtraces of the 9, 4.4 and 3 Kb species, the rest of the tissues testedmainly expressed the 4 Kb mRNA species. As expected from previousstudies for DOC1, I20 cDNA did not hybridize significantly to any mRNAspecies from the individual human cancer cell lines tested (MTN humancancer cell line blot from Clontech), thus confirming I-20 as beingencoded by a tumor suppressor gene.

Since the I-20 ORF contained no stop codon and extended 5′ past the ORFproposed for DOC1, we explored the possibility that in skeletal muscleI-20 represents a partial sequence of a larger protein. By probing thecorresponding cDNA library with the I-20 cDNA, we isolated andcharacterized by nucleotide sequencing four overlapping cDNA cloneswhich in total comprise an ORF encoding a predicted 764-amino acidpolypeptide of 90 kDa that was named GIP90 (SEQ ID NO:10), for GPBPinteracting protein 90 kDa. The existence of GIP90 mRNA was confirmed byisolating and nucleotide sequencing a continuous PCR fragment derivedfrom the same library containing the proposed overlapping ORF. The moreremarkable structural features of GIP90 are the presence of two nuclearlocalization signals (NLS), one in the N terminal region and another atthe C terminal region, and a highly predictable coiled-coil formationthrough most of its sequence including two leucine zippers.

Using the cDNA nucleotide sequence of GIP90 (“GIP90 cDNA”) (SEQ ID NO:9)we carried out a BLAST search against the human genome and found thatGIP90 cDNA matched at chromosome 3 (3q12) (genomic DNA accession numbersNT_(—)030634 for exon I and NT_(—)033050 for the rest of the exons). Wedetermined the exon/intron structure for the GIP90 genomic sequence,which encompass a total of six exons (FIG. 1). Exons I-IV of the GIP90gene contain 5′ untranslatable sequence and encode the first 201residues of an N-terminal segment of 240 residues that is absent in DOC1and DOC1-related protein (GenBank accession number AAH27860). Exon Vencodes the remaining 39 residues not present in DOC proteins as well asthe additional 524-residues of GIP90, and exon VI contains 3′untranslatable sequence.

Comparison of the GIP90 cDNA and the GIP90 genomic sequence revealed theexistence of an adenine (A) at position 2,720 (A²⁷²⁰) in the GIP90 cDNAthat was not present in the GIP90 genomic DNA, suggesting that GIP90cDNA represents either a cDNA artifact, or a native mRNA species thatderives from a DNA polymorphism or mRNA editing. Mutational artifactsare generally unique events unlikely to be found in more than one cDNAmolecular species. We have identified A²⁷²⁰ in at least two differentGIP90 cDNA fragments, representing two different reverse transcriptionevents, and PCR on total cDNA from the human muscle library (Clontech)using a forward primer from exon I and a reverse primer from exon VI,and subsequent direct sequencing, revealed that the resulting cDNAexclusively contained A²⁷²⁰. A homologous nucleotide was also found in aDOC1 encoding sequence, but not in DOC1-related protein encodingsequences. These results indicate that the A²⁷²⁰ in the GIP90 cDNA doesnot represent an artifact.

In order to further analyze the origin of GIP90 cDNA, we studied theexpression of GIP90 in two independent human skeletal muscle tissuesamples by RT-PCR. We were unable to amplify GIP90 mRNA from thesesamples. In contrast, we isolated and characterized a continuous cDNAfragment (SEQ ID NO:11) representing a related mRNA species that encodesa 130 kDa polypeptide (1135-residues) that we named GIP130a (SEQ IDNO:12). GIP130a results from faithful transcription and translation ofthe GIP90 genomic sequence (ie: no A²⁷²⁰), suggesting that a specificmechanism for mRNA diversification is responsible for the production ofGIP90 encoding mRNA from the GIP90 genomic sequence.

To further explore the mRNA diversification mechanism of theDOC1/GIP90/130 family, we compared the nucleotide sequences encodingDOC1/DOC1-related protein, GIP90, and GIP130a. Several nucleotidedifferences were identified, namely: (1) DOC-1 and DOC1-related mRNA aredevoid of exon I-IV; (2) DOC1 mRNA showed nucleotide deletions of 42-and 18-bp in exon V, and both DOC1 and DOC 1-related mRNA contain anadditional 276-bp at the 3′ end of this exon, which corresponds to anintron sequence in GIP90/130a; (3) DOC-1 and DOC1-related mRNAs are bothdevoid of exon VI.

Therefore, it appeared that the expression of exon VI is associated withexpression of GIP90/130a mRNAs, and that DOC-1 and DOC1-related mRNAsare exclusively encoded by an intron-extended exon V. The existence ofDOC-1 mRNAs containing exons I-IV was then assessed by PCR of MRNA fromhuman skeletal muscle and from human 293 cells. We obtained twodifferent cDNAs (SEQ ID NOS: 13 and 15) both containing exon I-Vsequences and DOC-1 exclusive exon V, and diverging with respect to eachother in one single nucleotide (A/G) at position 975, which leads to anamino acid change at position 168 (H¹⁶⁸/R¹⁶⁸). This results in twodifferent 1133-residue long polypeptides (130-kDa) which we namedGIP130b (SEQ ID NO:14) and GIP130c (SEQ ID NO: 16), respectively. Acomparison of the amino acid sequences of GP90/130 polypeptides and theDOC1 polypeptide family is shown in FIG. 3.

The amino acid sequence of rat filamin A-interacting protein (FILIP)(Genbank accession number BAC00851) and hypothetical human KIAA1275protein (Genbank accession number BAA86589) are highly homologous(approximately 50%) to the GIP90/130 and DOC proteins. This suggeststhat these genes are related and that FILIP, KIAA1275 and GIP90/130 arelikely to share biological functions. Therefore, knowing that FILIPimpairs cell migration of cortical neurons (Nature Cell Biology 2002July; 4(7):495-501), it is plausible to hypothesize that GIP90/130polypeptides exert their tumor suppressor activity, at least in part, byimpairing cell migration.

The above data demonstrate that the DOC-1/GIP90/130 mRNA family resultsfrom a complex diversification mechanism operating on the expression ofthe corresponding gene (GIP90 genomic sequence). Thus, we have foundthat the presence of R¹⁶⁸ or H¹⁶⁸ is the result of a GIP90 genomicsequence polymorphism. The presence of exon V, which is characteristicof GIP90/GIP130a (exon Va), is linked to the expression of exon VI andrepresents a complex alternative exon splicing in which the alternativeuse of two 5′ splice sites of an intron is coordinated with the splicingof an alternative 3′ terminal exon. Thus, when the more upstream 5′splice site is used to yield a shorter exon V (exon Va), the 3′ terminalexon (exon VI) is spliced, whereas when using the more downstream 5′splice site resulting in a larger exon V (exon Vb), the 3′ terminal exon(exon VI) is not spliced. Regarding A²⁷²⁰, we still are in the processof determining the specific diversification mechanism responsible forits presence. The exon/ intron structure of the gene for theDOC-1/GIP90/130 family is shown in FIG. 1 and a scheme for the morerelevant features regarding mRNA and protein structure for the GIPfamily is presented in FIG. 2. Finally, similar genetic diversificationmechanisms perhaps are responsible for the deletion of C²⁷⁰⁸¹ in DOC1and an aberrant alternative splicing within long exons (previouslydescribed for other genes) appears to account for the 42- and 18-bpdeletions found in DOC1 mRNA.

The presence of R¹⁶⁸ in GIP90 generates a putative bipartite NLS signaland a consensus for PKA phosphorylation, whereas the presence of A²⁷²⁰causes a frame-shift in the ORF encoding GIP90, which results in theappearance of a second nuclear localization signal and a premature stopcodon. The latter removes a total of 386 residues of the C terminalregion that is present in GIP130 proteins. These residues appear toconform to a domain with no predictable coiled-coils containing a numberof putative O-glycosylation sites (FIG. 2).

Characterization of GIP90/130 Interactions

Using a yeast two-hybrid system, we found that the four members of theGIP90/130 interact with GPBP, although to a more limited extent thanI-20 (SEQ ID NO:6). GIP90 displayed the strongest interaction with GPBP,whereas individual GIP130 proteins interacted similarly with GPBP,although to a lesser extent than GIP90. These data implicate theC-terminal residues of the GIP130 proteins, which are not present inGIP90, and also the C-terminal residues of GIP90 not present in I-20 ina negative modulation of the interaction of GIP90/130 polypeptides withGPBP. Deletion of the N terminal 240-residues of GIP90, GIP130b, andGIP130c resulted in molecular species that do not interact with GPBP,indicating that the N-terminal region contains residues involved in theinteraction of GIP90/130 polypeptides with GPBP. All of these findingsaccount for the observation that I-20 (SEQ ID NO:6), which contains thebulk of this N terminal region (residues 86-240), and does not harborthe inhibitory C terminal regions, displayed the strongest interactionin a two hybrid system with GPBP. The production of additional I-20deletion mutants and their use in specific two hybrid studies permittedthe identification of two specific regions of I-20 that are essentialfor GPBP interaction as well as the identification of other residuesdirectly involved but not essential for the interaction (FIG. 4).

GIP90/130 polypeptides self-aggregate and aggregate with each other in ayeast two-hybrid assays, indicating that, similarly to GPBP (WO00/50607), GIP90/130 polypeptides aggregate to form homo and heterooligomers. No significant differences were found among GIP90/130 fulllength polypeptides in their ability to self-aggregate. Deletion of theN-terminal 240-residues from GIP130b/c results in DOC1-related protein,which aggregates more efficiently and does not interact with GPBP. Sincethe deleted residues contain motifs for I-20 self-aggregation, it isconceivable that the deleted region contains residues that are criticalfor GIP90/130 aggregation, but not for DOC/DOC1-related proteinaggregation, and that GIP90/130 polypeptides and DOC1 polypeptidesaggregate in a different manner. Since the N terminal 240 residues alsocontain essential residues for GIP90/130 polypeptide interactions withGPBP, this further suggests that GPBP interaction negatively modulatesGIP90/130 polypeptide aggregation but not DOC aggregation. Consistently,two hybrid assays using I-20 deletion mutants show that essentialsequences for GIP90/130 interactions with GPBP and for I-20 aggregationoverlap extensively (FIG. 4), strongly suggesting that GPBP binding toGIP90/130 polypeptides prevents GIP90/130 polypeptide aggregation butnot DOC aggregation. Accordingly, we have observed with a yeastthree-hybrid system that GPBP expression efficiently impairs both I-20and GIP90 aggregation, and that I-20 and GIP90 efficiently impair GPBPaggregation.

Deletion mutants were obtained using specific primers and PCR, followedby cloning of the resulting cDNAs in the pGBT9 and pGAD424 vectors. Theassays were performed in SFY526 or HF7c Saccharomyces cerevisiaestrains, with pGBT9 as GAL4 binding domain vector and pGAD424 as GAL4activation domain vector, by the lift colony assay procedure. Briefly,the yeast cells were co-transformed with constructs of both bindingdomain and activation domain vectors, and the co-transformants wereselected in medium deficient in both tryptophan and leucine. After fivedays of incubation at 30° C. the colonies were tested for the expressionof β-galactosidase with X-Gal substrate (0.75 mg/ml). The intensity ofthe blue color displayed in the assay informed us about the relativestrength of the interactions. When the assays were performed with theHF7c strain, the interactions were assessed by the lift colony assayprocedure and by growth in medium deficient in histidine, tryptophan andleucine. For yeast three-hybrid system, we used the pBRIDGE vector,which allows the conditional expression of a third protein apart fromthe usual GAL4 binding and activation domain-fusion proteins of thetwo-hybrid system. In this case, the expression of GPBP or I-20 or GIP90was driven by Met25 promoter, active in absence of methionine. In theseexperiments, the transformed SFY526 cells were plated in mediumdeficient in tryptophan, leucine and methionine, and subjected to thecolony lift assay after five days at 30° C. In the case of the strainHF7c the colonies grown in the cited plates were streaked on medium withthe additional deficiency of histidine.

In an attempt to establish the viability of these molecular interactionsin human cells, the interaction between GIP90 and GPBP was assessed in amammalian two-hybrid system using 293 cells. We used the CLONTECHmammalian two hybrid kit, with vectors pM and pRK5-GAL4BD as GAL4binding domain vectors and pVP16 as activation domain vector. Wetransfected 293 cells by the calcium phosphate procedure with theappropriate constructs and reporter vectors and the interactionsdetermined by the CAT ELISA kit (Roche), following the manufacturer'sinstructions.

Finally, using a yeast two hybrid system, we investigated theinteractions between pol κ/pol κ76 and GPBP/GPBPΔ26 and we got nopositive results. However, when we challenged interaction between polκor pol κ76 and I-20, we obtained positive results with pol κ76 but notwith pol κ. The positive interaction of I-20 with pol κ76 suggests thatGIP90 is a biological bridge between GPBP and pol κ76 and that the threeproteins are partners in specific strategies which become deregulatedduring autoimmune pathogenesis.

From all these data, we conclude that: (1) GIP90/130 polypeptidesaggregate in a different manner than DOC/DOC 1-related polypeptides; (2)GPBP interacts with GIP90/130 polypeptides and this interactioncounteracts GIP90/130 polypeptide aggregation; (3) GPBP does notinteract with DOC/DOC1-related proteins, and therefore GPBP is notexpected to influence DOC/DOC1-related protein aggregation; (4) I-20contains essential amino acid sequences involved in GPBP interactionwith GIP90/130 polypeptides and in GIP90/130 polypeptide aggregation;(5) the C terminal domain of GIP130 species exerts a negative effect ontheir interactions with GPBP, and (6) GIP90/130 polypeptides containsequences not present in I-20 that negatively modulate both GIP90/130polypeptide interaction with GPBP and GIP90/130 polypeptide aggregation.

Further Characterization of GIP90/130

Given that GPBP is a protein kinase, we assessed the capacity of GPBP tophosphorylate GIP90 in vitro by using purified yeast recombinantcounterparts. GIP90 was cloned in pHIL-D2 vector in frame with the FLAGtag at N-terminal position and with a 6 histidine tail at C-terminalposition. It was expressed in the Pichia pastoris expression system(Invitrogen) and purified with an affinity resin (Clontech) makingprofit of the polyhistidine tail, using an 8 M urea-containing breakingbuffer, which was eliminated by dialysis against Tris-buffered saline.The purified protein was incubated with yeast recombinant GPBP in asuitable reaction buffer and labelled for 12 hours at 30° C. Thephosphorylation mixtures were analysed by Western blot usingFLAG-specific antibodies (Sigma) and autoradiography. Incubation ofpurified GIP90 and GPBP in the presence of [γ³²P] ATP resulted in ³²Pincorporation into GIP90, thus confirming that GPBP interacts with GIP90and phosphorylates it.

Remarkable structural features of GIP90/130 proteins are (1) theexistence of two nuclear localization sequences (NLS) whose presenceappears to be regulated by single nucleotide replacement or addition(see above); and (2) the existence of a large number of predictablecoiled-coil motifs including two leucine zippers. Consequently we haveassayed the ability of GIP90/130 and DOC1-related protein to inducetranscription from a heterologous promoter of a reporter gene. This wasaccomplished by fusing either GIP90, GIP130a, GIP130b or DOC1-relatedprotein to the binding domain of GAL4 transcription factor in a highlevel expression pAS2-1 vector (Clontech) and transforming SFY526 yeastcells carrying a LacZ reporter gene under the control of a promoter witha GAL4 binding site. Transformants were selected in tryptophan-deficientmedium at 30° C. for five days and colony lift assays performed. TheGIP90, GIP130a, and GIP130b fusion polypeptides, but not DOC1-relatedprotein fusion polypeptides, efficiently induced expression of LacZ, asestimated by the appearance of β-galactosidase activity.

We have also expressed GIP90 in bacteria, and have used thecorresponding recombinant protein to immunize both rabbits and mice toobtain respectively polyclonal and monoclonal antibodies specific forGIP proteins. GIP90 was cloned in pGEX vector, in frame withglutathione-S-transpherase cDNA. The resulting construct was used totransform DH5α cells and expression of the GST-GIP90 fusion protein wasinduced with IPTG and further purified on glutathione affinity column.GST-GIP90 purified protein was used to immunize both rabbits and mice inorder to obtain respectively polyclonal and monoclonal antibodies. Theseantibodies were used to identify a native protein in 293 cellsdisplaying the same mobility as recombinant GIP130 which likelyrepresents endogenous GIP130b or GIP130c, since exon VI appears to notbe expressed in these cells, as determined by specific RT-PCRapproaches. One of the monoclonal antibodies (Mab3) maps in the Nterminal 240 residues of GIP90, whereas Mab 8 maps within the next 509residues (i.e.: between residues 241-750).

By indirect immunofluorescence on COS-7 cells transiently expressingrecombinant GIP90 we have identified cells that expressed GIP90 in thenucleus, cells expressing GIP90 in the cytosol, and cells that expressedGIP90 in both the nucleus and the cytosol. When these cells co-expressedrecombinant GIP90 and GPBP, double indirect immunofluorescence revealedexpression of the two proteins at the cytosol and in some cells GIP90was also detected in the nucleus. We have not seen GIP90 and GPBP beingco-expressed in the nucleus. Finally, using confocal microscopy andNIH3T3 or 293 cells, we have confirmed nuclear localization of GIP90 andcytosolic co-localization GIP90/GPBP. These cells do not expressdetectable levels of GIP90/130 polypeptides, as no significantfluorescence was detected when non-transfected cells were incubated withanti-GIP antibodies and an appropriate secondary antibody. Forimmunofluorescence and confocal microscopy studies, GIP90 cDNA wascloned in pRK5 mammalian expression vector, and this construct was usedalone or co-transfected with GPBP cloned in pCDNA3 vector (Invitrogen),using the DEAE-dextran or calcium phosphate procedures. After 24 hoursof incubation at 37° C., the cells were washed with phosphate-bufferedsaline (PBS), fixed with methanol or methanol:acetone, blocked with 3%BSA in PBS and incubated with a pool of mouse anti-GIP90 monoclonalantibodies and rabbit anti-GPBP polyclonal antibodies. FITC-conjugatedanti-mouse IgG and TRITC-conjugated anti-rabbit IgG antibodies wererespectively used as secondary antibody.

Finally, we have performed immunohistochemistry studies on paraffinembedded human tissues and have found GIP proteins to localize in anumber of cells and structures also expressing GPBP.Immunohistochemistry studies were done on human multi-tissue controlslides (Biomeda, Dako), using the ABC peroxidase method. GIP proteinsare widely expressed in human tissues, but are more abundantly expressedin some locations. A strong staining is found in smooth muscle cells,particularly in those of vessel walls, with a diffuse cytoplasmicpattern. There is intense expression in alveolar septa, with a linearpattern suggestive of being associated to basement membrane locations,along with cytoplasmic staining of the pneumocytes. The kidneys showexpression in the epithelial cells of the tubules, mainly in distantones, and also in mesangial cells and podocytes of the glomerulus. Inthe pancreas there is staining in the cells of endocrine Langerhansislets. In the adrenal gland, the cortical cells show higher expressionthan the medullar cells. In the liver, hepatocytes show expression ofthe GIP90/130, which is higher at the epithelial cells of the biliaryducts. The white matter of the central nervous system shows diffusestaining with a fibrillar pattern, with presence also found in someneuronal bodies. Expression of the GIP90/130 is also evident at theepithelial cells of the prostate, breast, bronchi and intestine, instriated muscle cells of the myocardium, in secretory cells of thepituitary, and in spermatogonium and Leydig cells in the testicle.

The expression of the GIP90/130 is quite similar to that previouslydescribed for GPBP (WO 00/50607), with staining in tissues targeted byautoimmune responses, such as the Langerhans islets (type I diabetes),the white matter of the central nervous system (multiple sclerosis), thebiliary ducts (primary biliary cirrhosis), the cortex of the adrenalgland (Addison disease), alveolar septa (Goodpasture syndrome), andspermatogonium (male infertility).

The evidence suggests that GIP90/130 is a family of proteins encoded bya tumor suppressor gene, which display transcription factor activity,and which interact and are phosphorylated by GPBP. Given the role ofGPBP in autoimmune pathogenesis and in cancer, GIP90/130 represent apotential therapeutic or therapeutic target in these disorders.

1. An isolated antibody directed against a polypeptide with the aminoacid sequence of SEQ ID NO:4.
 2. The isolated antibody of claim 1,wherein the antibody is a monoclonal antibody.
 3. The isolated antibodyof claim 1, wherein the antibody is a polyclonal antibody.
 4. Apharmaceutical composition comprising: a) an isolated antibody accordingto claim 1; and b) a pharmaceutically acceptable carrier.
 5. Thepharmaceutical composition of claim 4, wherein the antibody is amonoclonal antibody.
 6. The pharmaceutical composition of claim 4,wherein the antibody is a polyclonal antibody.